Thesis for the Master’s degree in Molecular Biosciences
Main field of study in Molecular Biology
Development of a reporter gene construct that can be
used for the localization of RNA pyrophosphohydrolase
(rppH) in cells of Chlamydomonas reinhardtii cells.
Biruk Luelseged
60 study points
Department of Molecular Biosciences
Faculty of mathematics and natural sciences
UNIVERSITY OF OSLO
December/2012
Acknowledgement
This work was carried out during the period of May 2012 - November 2012 at the University
of Oslo, department of Molecular Biosciences.
I would like to thank Professor Uwe Klein for his guidance and assistance in writing of this
thesis.
I would also like to thank the lab members for nice time.
Finally I would like to thank Beate, Reier, Erland and Anja for their financial support, and
constant heartfelt encouragement.
Oslo, December 2012
Biruk Luelseged
Abstract
Abstract
The concept that messenger RNA (mRNA) degradation in E. coli begins with endonucleolytic
cleavage has been challenged by the recent discovery that the conversion of the 5’ terminus
from a triphosphate to a monophosphate is required prior to endonucleolytic activity. RNA
pyrophosphohydrolase (RppH) initiates the degradation of transcripts by removing
pyrophosphate from the 5'-end of mRNAs which allows binding of RNase E/ RNase J in
bacteria. A putative RNA pyrophosphorylase is present in cells of the unicellular green alga
Chlamydomonas reinhardtii and several lines of evidence suggest that the protein is involved
in mRNA degradation in the chloroplast. In order to determine the location of the protein in
Chlamydomonas cells, the 5’ region of the rppH gene was tagged to a codon optimized green
fluorescent protein and introduced into Chlamydomonas cells.
This study focuses on developing a reporter vector construct that can be used to transform the
Chlamydomonas reinhardtii nuclear genome. GFP (Zsgreen 1) was codon optimized by using
a Codon Usage Database. The reading frame of the optimized synthetic GFP was adjusted and
cloned into the pBluescript-5’rppH SK+ vector. The 5’rppH-GFP gene fragment was then
sequentially cloned into the pE coli-Cterm 6HN protein expression vector, intermediate vector
and finally in the pChlami transformation vector. All constructs were verified by restriction
cutting and sequencing. Transformants were screened for the presence of the chimeric GFP
gene by PCR. The screening experiments have led us to choose one transformant that could
be used for further work. In addition although fluorescence was not checked, the optimized
synthetic GFP was expressed in E. coli (BL21DE3) cells confirming the functionality of the
synthetic GFP construct in vivo.
i
Table of Contents
Abstract ....................................................................................................................................... i
1.
INTRODUCTION .............................................................................................................. 1
1.1
Chlamydomonas reinhardtii ........................................................................................ 1
1.2
Genetics and Chlamydomonas..................................................................................... 1
1.3
Organelle Gene regulation ........................................................................................... 2
1.3.1
1.4
Anterograde/Retrograde signaling ....................................................................... 2
Chloroplast mRNA Turnover ...................................................................................... 3
1.4.1
Cis acting elements............................................................................................... 5
1.4.2
Trans acting factors .............................................................................................. 5
1.4.3
Effect of redox state ............................................................................................. 6
1.4.4
Ribonuclease susceptibility .................................................................................. 6
1.5
Bacterial gene regulation ............................................................................................. 7
1.6
mRNA decay in bacteria .............................................................................................. 7
1.6.1
Bacterial mRNA stability ..................................................................................... 7
1.6.2
Bacterial mRNA processing and degradation ...................................................... 8
1.7
Nudix Hydrolase .......................................................................................................... 9
1.8
RNA pyrophosphohydrolase (RppH) .......................................................................... 9
1.9
Sub-cellular localization ............................................................................................ 10
1.10 GFP ............................................................................................................................ 10
1.11 Aim of the project ...................................................................................................... 12
2.
Materials and methods ...................................................................................................... 13
2.1
PCR ............................................................................................................................ 13
2.2
Construction of fusion proteins and vectors .............................................................. 13
2.3
Agarose gel electrophoresis ....................................................................................... 14
2.4
Ethanol precipitation.................................................................................................. 14
2.5
Bacterial cells ............................................................................................................ 14
2.6
Transformation of Chlamydomonas .......................................................................... 14
2.7
Selection of transformants ......................................................................................... 15
2.8
Cell culture ................................................................................................................ 15
2.9
Selective marker ........................................................................................................ 15
2.10 Codon optimization ................................................................................................... 15
2.11 Screening of transformants with PCR ....................................................................... 16
2.12 Recombinant protein expression in E. coli (BL21DE3) ............................................ 16
2.12.1
Purification of Histidine-tagged GFP ................................................................. 17
2.13 SDS PAGE ................................................................................................................ 17
3.
Results ............................................................................................................................... 18
3.1
Optimization of GFP ................................................................................................. 18
3.2
Cloning ...................................................................................................................... 20
3.2.1
pBluescript II SK+ 5’ rppH-GFP vector: Cloning step A ................................. 21
3.2.2
pE coli-C-term-5’rppH-GFP 6×HN vector: Cloning step B ............................. 23
3.2.3
Intermediate 5’ rppH-GFP-6×HN vector: Cloning step C ................................ 24
3.2.4
Transformation vector pChlami-5’rppH-GFP-6×HN: Cloning step D ............. 26
3.3
Transformation and selection of transformants ......................................................... 27
3.4
Analyses of transformants by PCR ............................................................................ 28
3.5
Recombinant protein expression and purification ..................................................... 32
4.
Discussion ......................................................................................................................... 34
5.
Conclusion ........................................................................................................................ 37
References ................................................................................................................................. A
APPENDIX I .................................................................................................................................. I
APPENDIX II .............................................................................................................................. II
APPENDIX III ............................................................................................................................ III
APPENDIX IV ............................................................................................................................ VI
APPENDIX V ............................................................................................................................VII
APPENDIX VI ......................................................................................................................... VIII
APPENDIX VII .......................................................................................................................... IX
Introduction
1. INTRODUCTION
1.1 Chlamydomonas reinhardtii
Chlamydomonas is a genus of unicellular green alga. Morphologically the cells are spherical
or ellipsoidal with a size of ~ 10 µm in diameter and have two flagella of the same size that
are situated at the anterior end of the cell. They are found mostly in fresh water, on damp soil,
and there is a report that few occur also in the sea.
Chlamydomonas reinhardtii is one of the most widely used model organisms. Cells have the
ability to grow quickly with a generation time of approximately 5 hours. They are haploid and
can reproduce asexually or sexually.
It has been used as a model organism for photosynthesis and chloroplast biogenesis studies,
mitochondrial biogenesis, gametogenesis and mating, assembly of flagella and motility, and
cellular metabolism. It is an excellent system to study mutations as it has only a single copy of
each gene and chloroplast, mitochondrial and nuclear genomes have been sequenced (Maul et
al. 2002, Merchant et al. 2007, Smith and Lee 2008). Cells have the capacity to grow with
light as sole energy source or on acetate in darkness, facilitating detailed examination of genes
and proteins critical for photosynthetic or respiratory function. This haploid organism grows
rapidly on both solid and liquid medium (Grossman et al. 2003).
1.2 Genetics and Chlamydomonas
Whole-genome sequencing provides a platform for identification of mutations in
Chlamydomonas (Dutcher et al. 2012). Besides this, C. reinhardtii is one of the best studied
alga in circadian rhythm research (Matsuo et al. 2008) which are biological rhythms that
continue under constant conditions of light and temperature with a period of about 24 hour
(Schulze et al. 2010). Biogenesis and the mode of action of miRNAs and siRNAs were also
studied in C. reinhardtii (Ibrahim et al. 2010). Based on the degree of compelimentary of their
small ~ 21 nt sequence and their target mRNA, miRNAs exert or regulate gene expression by
cleaving or degrading mRNAs. The degradation effect of these miRNAs is highly observed in
plants, in contrast animal miRNA are involved in translational repression. Despite the whole
genome complexity differences between those of higher organisms and Chlamydomonas cells,
the unicellular algal genome has been shown to constitute many of the small RNAs. Strikingly
the miRNAs identified in C. reinhardtii share structural and functional homology with both
plant and animal miRNAs (Zhao et al. 2007).
Based on the completed annotation of the C. reinhardtii genome, micro array platform was
designed to validate the function of genes that are related to efficient light harvesting and
photosynthetic electron transport (Toepel et al. 2011). Because chlorophyll synthesis also
occurs in the dark in C. reinhardtii, the photosynthetic apparatus can be assembled in the
1
Introduction
absence of light. Therefore it is possible to study photosynthetic reactions in light-sensitive
mutants (Rochaix 2002, Nishimura 2010).
It is amenable to a diversity of genetic and molecular manipulations. In addition engineering
of the plastid genome was first undergone in C. reinhardtii (Boynton et al. 1988). The recent
sequencing of the nuclear genome and the availability of numerous molecular tools including
transformation of the three (nuclear, plastid and mitochondrial) genomes, makes C.
reinhardtii an attractive model for molecular investigations.
1.3 Organelle Gene regulation
Chloroplast genomes encode the most important genes for photosynthesis (Maliga and Bock
2011). The genome has uniform densities of genes, simple sequence repeats and transposable
elements (Merchant et al. 2007). Chloroplast gene expression in C. reinhardtii is a complex
process that can be influenced by mRNA processing, mRNA stability and protein turnover
(Rasala et al. 2011). But unlike nuclear genes, genes encoded by chloroplast are not affected
by transcriptional silencing and position effects (Michelet et al. 2011). The Chlamydomonas
chloroplast genome has only a few introns located in the psbA, psaA, and 23S rRNA genes
(Herrin and Nickelsen 2004).
Studies on higher plants have shown that there are two RNAPs (RNA polymerases), NEP
(Nuclear encoded RNA polymerase) and PEP (Plastid encoded RNA polymerase). PEP has a
catalytic core consisting of α, β, β´ and β´´ subunits which is thought to require σ70-like
factors that specifies initiation of transcription downstream of promoter sequences which
resemble bacterial 10/35 promoters (consensus: -10 TATAAT,- 35 TTGACA). Chloroplast
gene expression is also dependent on factors that are encoded by the NEP. Characterizing this
polymerase has revealed that it is a single catalytic subunit polymerase homologous to the
T7/T3 phage polymerases. Unlike those of higher plants, Chlamydomonas lacks NEP. A
recent study has shown that C. reinhardtii expresses a single σ70-like factor which likely
functions in chloroplast transcription (Bohne et al. 2006).
1.3.1 Anterograde/Retrograde signaling
The chloroplast genome encodes proteins that are involved in transcriptional and translational
apparatus. Concentration of chloroplast proteins and their function is dependent on the
anterograde (nucleus to chloroplast) and retrograde (chloroplast to nucleus) signaling
mechanisms. Anterograde mechanisms coordinate gene expression in organelles in response
to endogenous and environmental stimuli that are perceived by the nucleus. Retrograde
mechanisms transmit signals that originate in the organelles to regulate nuclear gene
expression, which can then alter anterograde control (Woodson and Chory 2008).
The continuous flux of proteins into and out of the nucleus as well as among intra-nuclear
compartments controls central events such as DNA replication and mRNA processing (Gorski
et al. 2006). In C. reinhardtii many DNA-binding proteins directly or indirectly involved in
the regulation of gene expression have been shown to be at least transiently located in the
2
Introduction
nucleus (Winck et al. 2012). Nucleus encoded proteins in Chlamydomonas are the basis for
anterograde signalling that enables the nucleo-cytoplasmic compartment to control chloroplast
gene expression (Michelet et al. 2011).
The nuclear genome of C. reinhardtii is 100 to 110 million bp, comprising 17 genetic linkage
groups (Kathir et al. 2003) presumabily corresponding to 17 chromosomes (Merchant et al.
2007), with a very high GC content (nearly 65%). Chlamydomonas nuclear genome is
haploid, in contrast to approximately 80 copies of the chloroplast genome (Eberhard et al.
2011). The number of selection markers available for genetic engineering in Chlamydomonas
is relatively small compared to those for other model systems. Foreign genetic material
introduced to C. reinhardtii nuclear genome recombines randomly at any site in its genome
(Kindle et al. 1989), which may result in deletion of functionally important genes. Moreover
transgene expression level is very low even if the transformation reaction is highly efficient
due to the following possible reasons; the first being inadquate amount of promoter and
regulatory regions affecting the transcription level of the gene (Leon-Banares et al. 2004) of
interest. In addition epigenetic silencing of transgenes, especially single copy transgenes can
be transcriptionally silenced without detectable cytosine methylation (Jeong et al. 2002). The
other being due to codon usage bias; codon frequently used in certain organisms is rarely used
in others (Nakamura et al. 2000). This has hampered the applicability of C. reinhardtii for
biotechnological expression analyses. Recently new genetically engineered resistance markers
were developed (Meslet-Cladiere and Vallon 2011).
1.4 Chloroplast mRNA Turnover
The level of mRNA within a cell depends on the balance between transcription and
degradation. mRNA is the most varied class of RNA with respect to its size and stability. The
activity of RNA is determined by its structure (Zuker et al. 1999). As such the stability of a
given mRNA transcript can be determined by the presence of sequences within the 5’ UTR
(untranslated region) (Salvador et al. 2004), which can be bound by trans-acting RNA-binding
proteins to inhibit or enhance mRNA degradation (Hollams et al. 2002). In addition the
structural characteristics of 5' untranslated region, such as length, the presence of AUG
upstream of the initiator greatly affect the efficiency of the translational process (Pesole et al.
2000) and thereby the longevity of the transcript. Moreover sequences that are found in the
protein coding region have been shown to control promoter activity in Chlamydomonas
chloroplast rbcL gene (Klein et al. 1994).
Regulated mRNA stability is achieved through fluctuations in half-lives in response to
environmental stimuli like nutrient levels and temperature shifts (Guhaniyogi and Brewer
2001). In bacteria for example, mRNA half-lives can be in seconds or in hours, whereas in
vertebrates they range from minutes to days. The half-lives of stable RNAs approach or even
exceed the doubling times of the cells in which they are made (Meyer et al. 2004); and thus
their concentration does not depend on cell growth.
mRNA turnover implys rapid synthesis and equally rapid destruction (Meyer et al. 2004).
Selective degradation of messenger RNAs enables cells to regulate the levels of particular
3
Introduction
mRNAs in response to changes in the environment (Mackie 1998). The current knowledge of
chloroplast mRNA degradation begins by endonucleolytic cleavage which is followed by 3’
polyadenylation of the cleavage products. Addition of poly A rich sequences to the
endonucleolytic cleavage products of mRNA is required to target these molecules for rapid
exonucleolytic degradation (Schuster et al. 1999). The polyadenylated cleavage products are
subsequently degraded by 3’-5’ exonuclease. The RNA hairpins (secondary structures) that
are found at the 3’ termini impede the 3’-5’ exonucleases, thereby stabilizing upstream RNA
(Pfalz et al. 2009). Essential factors that control mRNA turnover in chloroplast such as cis
elements, proteins that bind to them, ribonuclease susceptibility, and redox status are briefly
discussed below.
Figure 1.1. Model for chloroplast mRNA degaradation. Endonuclease cleavage is initiated by the
RNase J and RNase E enzymes. The polynucleotide tail addition is facilitated by PNPase or PAP. The
RNA fragment where the 3’ end is blocked by a PPR protein requires a 5’→3’ decay. The absence or
low frequency of secondary structures at the 3’ end can also mediate a 3’→5’ decay by PNPase or
RNase II. Image modified from (Stern et al. 2010).
4
Introduction
1.4.1 Cis acting elements
The function of RNA can only be understood in terms of its secondary or tertiary structure
(Zuker et al. 1999). Cis regulatory elements on chloroplast RNA are regions that are required
for its stability or its degradation. They are usually found on the 5’ and 3’ UTR of mRNA.
Sequences within the 5'-UTR are essential for translation (Anthonisen et al. 2001). The 5’
UTR have been shown to regulate the translation of the psbC (Zerges et al. 1997), psbA
(Trebitsh et al. 2000), psbD (Nickelsen et al. 1999) genes. In addition inverted repeat
structures which are nucleotide sequences that are the reverse complement of another
sequence downstream have been found in chloroplast genome of all Chlamydomonas species
examined (Harris 2001). These inverted repeats have been shown to determine the stability of
chloroplast rbcL mRNA in C. reinhardtii (Goldschmidt-Clermont et al. 2008). Sequence and
condition-dependent 5′→3′ mRNA-degradation pathway was also shown in the chloroplast of
C. reinhardtii (Salvador et al. 2011). The requirement of specific 5’ sequence for RNA
longevity is also determined by an interaction of this element with a trans-acting factor (Suay
et al. 2005).
1.4.2 Trans acting factors
Trans acting factors are proteins that are mostly nuclear encoded. They are synthesized in the
cytosol and subsequently imported into the chloroplast, where they interact with their target
sites on either chloroplast RNAs or proteins (Nickelsen et al. 1999).
Several protein families playing essential roles in the mRNA metabolism in chloroplast are
characterized by the occurrence of tandem repeat motifs. PPR members of the
pentatricopeptide repeat proteins function in RNA processing, and translation (Johnson et al.
2010, Stern et al. 2010). PPR family, which comprise of 35 amino acid repeats, primarily
interact with specific RNA-targets. 11 PPR protein-encoding genes have been identified in C.
reinhardtii (UniProt). The stabilizing mechanism of these PPR protein is by giving a shelter
to a specific region of RNA from nucleases, in the manner of a protein cap (Johnson et al.
2010). Functionally they were also implicated in protecting internal transcript sequence
elements (Zhelyazkova et al. 2012). Moreover in C. reinhardtii, a PPR protein is required for
stabilization of the rbcL mRNA (Johnson et al. 2010).
The tetratricopeptide repeat (TPR) is a degenerate 34-amino acid repeat motif, protein-protein
interaction module found in multiple copies in a number of functionally different proteins
(Blatch and Lassle 1999). For instance, Raa 1 with five copies of octatricopeptide repeat is
thought to improve the efficiency of psaA mRNA maturation in C. reinhardtii chloroplasts
(Merendino et al. 2006). NAC2 is also involved in stabilization of psbD transcripts (Boudreau
et al. 2000).
5
Introduction
Figure 1.2. Model for post-transcriptional modifications in chloroplasts. Various nuclear encoded
RNA-binding proteins participate in RNA processing events by binding directly to their target RNAs.
These events involve several steps, such as splicing and stabilization which control chloroplast gene
expression (Jacobs and Kuck 2011).
1.4.3 Effect of redox state
During photosynthesis, the source of electron is water. In the photosynthetic machinery,
different components are involved in redox reactions. Changes in the reduction/oxidation state
of these components are used as signals in gene regulation (Pfannschmidt et al. 2003). Light
influences chloroplast gene expression, RNA processing, transcript stability, translation and
turnover of proteins (Salvador and Klein 1999). In C. reinhardtii, the redox state regulates the
degradation of chloroplast transcripts (Salvador and Klein 1999).
1.4.4 Ribonuclease susceptibility
As has been mentioned, the extent to which mRNA is susceptible to degradation by
ribonucleases is dependent on its secondary structure. Thus secondary structures are important
to prevent mRNA from degradation. Moreover the binding of trans-acting proteins to a target
mRNA sequence can change its susceptibility to ribonucleases thereby promoting mRNA
degradation. These ribonucleases determine the longevity of mRNA in prokaryotes such as
bacteria.
Chloroplast mRNA and that of prokaryotic have certain differences. The distinguishing
features are the predominance of introns and complex patterns of processing from
polycistronic precursors (Stern et al. 2010). In addition, polyadenylation is associated with
RNA instability in prokaryotic cells, whereas in chloroplast nucleus-encoded mRNAs,
polyadenylation is to enhance their stability and promote translational initiation (Komine et al.
2000). Moreover the poly (A) tail sequences are composed of clusters of adenosines mostly
bound by guanosines, and on rare occasions, by cytidines and uridines (Schuster et al. 1999)
whereas in bacteria, the sequences are composed of clusters of adenosine residues.
6
Introduction
But the molecular biology of plastids is basically prokaryotic (Salvador et al. 2011).
Sequences composed of -35 and -10 elements resembling promoters of E. coli genes direct
transcription in vitro as chloroplast promoters in vivo (Blowers et al. 1990). Translation
regulation in chloroplasts has been also reported to be very similar to prokaryotes (Kozak
2005). The poly (A) tails found in Chlamydomonas chloroplasts are similar in length to those
of E. coli, being mostly between 20 and 50 nt (Komine et al. 2000). Chloroplast ribosomes
and that of bacteria share similarities on the mechanism of protein synthesis initiation and
inhibition (Ellis 1970). In addition higher plants plastid genome encodes subunits of an E.
coli-like RNA polymerase (PEP) which initiates transcription from E. coli σ70-type promoters
(Hajdukiewicz et al. 1997). Furthermore the ribonucleases involved in chloroplast mRNA
turnover are derived from bacteria (Barkan 2011).
1.5 Bacterial gene regulation
RNA degradation is a major component of RNA metabolism in determining its intracellular
levels during which rapid decay serves as a mechanism to adjust to environmental change
(Deutscher 2006).
Most of the mRNA found in bacteria is polycistronic where its genetic information is
translated to several polypeptides that are functionally related to each other. The genetic
information consists of functional gene clusters, termed as operon. Bacterial operons share
similarity wth the polycistronic transcription units of the chloroplast genome (Barkan 2011).
1.6 mRNA decay in bacteria
RNA stability, degradation and processing is determined by ribonucleases in bacteria. There
are two main classes of ribonucleases; endoribonuclease and exoribonuclease.
Endoribonuclease cleave RNA segment at the internal phosphodiester bonds. Whereas the
exoribonucleases cleave at the end of RNA chain. Exoribonucleases can act either on the 5’
end or 3’ end of an RNA segment.
1.6.1
Bacterial mRNA stability
Stabilizing elements such as the 5’-triphosphate of primary transcripts and strong secondary
structures are crucial in bacterial mRNA stability (Evguenieva-Hackenberg and Klug 2009,
Evguenieva-Hackenberg and Klug 2011). 5' secondary stem-loop structurs are able to protect
mRNA from attack by cellular ribonuclease that initiates mRNA degradation (Emory et al.
1992, Sharp and Bechhofer 2005). Ribosome binding at Shine–Dalgarno elements adjacent to
the translation initiation codon (collectively called ribosome binding site) also serves to
protect specific endonucleolytic target sites required for the initiation of decay (Petersen
1992).
7
Introduction
1.6.2 Bacterial mRNA processing and degradation
RNA processing and degradation determine transcript accumulation and thus are key
processes in the control of gene expression. RNA processing is used to describe RNasecatalyzed events leading to the generation of a functional RNA, while RNA degradation is
used to describe its decay, in which RNA processing leads to an increase in RNA half-life
relative to the primary transcript whereas RNA degradation leads to a decrease in half life.
RNA processing is relevant in the 5’ maturation of transcripts. The mRNA processing
depends usually, but not always, on the activity of RNases (Lehnik-Habrink et al. 2012).
RNA turnover in E. coli requires pathways that involve endoribonucleases and
3’‐exoribonucleases that modify RNA or affect its conformation (Carpousis et al. 2009). The
decay of most mRNAs in E. coli is initiated by RNase E, a 1061-amino acid enzyme which is
specific for single stranded regions in mRNA. The endoribonuclease RNase E is conserved
among proteobacteria and is essential for viability due to its role in turnover of mRNA in E.
coli (Anupama et al. 2011) where it acts on mRNA and small non-coding RNA. RNase E is
organized into a number of catalytic core domains that are arranged to different sub-domains
and has a long non-catalytic C-terminal extension (Carpousis 2007). The larger subdomain is
composed of RNA binding domain and 5’ sensor. Previous study (Garrey et al. 2009) where
the RNA binding subdomain and the 5′ sensor interact to form a certain compartment that
enhances 5’monophosphate binding was shown to have increased the RNase E activity.
RNase E is important in mRNA decay which cleave single stranded AU rich RNA (Mudd et
al. 1990). A 5’ end-independent mRNA decay pathway which requires endonuclease activity
is also reported (Deikus and Bechhofer 2011).
Exoribonucleases can act on mRNA either in the 5’→3’ or 3’→5’ direction in bacteria. Some
are involed in the maturation and stability of mRNA for instance, RNase J1 (Mathy et al.
2007) whereas others are involved in quality control, or degradation of defective RNAs such
as RNase R (Vincent and Deutscher 2009), PNPase or RNase II. E. coli cells contain multiple
3’→5’ acting exoribonucleases (Arraiano et al. 2010). The 3’ end of bacterial mRNAs is
protected from 3’-to-5’ exonuclease attack by secondary structure (Condon 2007). Thus, the
action of endonucleases generate new unprotected 3’ ends that are rapidly degraded by
exonucleases. Hence since E. coli lacks 5’ exoribonucleases (Kaberdin et al. 2011), it is
believed that endonucleolytic cleavage generally precedes 3′ exonucleolytic degradation
(Belasco 2010).
Similarly stem loop (hairpin loop) which is the building block for a secondary structure, at the
5’ end of E. coli transcripts can inhibit mRNA degradation by RNase E (Mackie 2000).
RNase E endoribonucleolytic activity is increased by the 5’ terminus state of its target RNA.
mRNA stabilization by a 5’-terminal stem-loop has also been reported in B.subtilis
(Hambraeus et al. 2002). Similar to E. coli, the presence of a 5′-triphosphate have stabilizing
effects on the downstream genes in B.subtilis (Lehnik-Habrink et al. 2012). In both organisms
RNA degradation begins with the conversion of the 5’-terminal triphosphate to a
monophosphate. The hydrolysis of a variety of nucleoside triphosphates is catalyzed in vivo
by the Nudix hydrolases.
8
Introduction
1.7 Nudix Hydrolase
The terminal RNA sequences are modified by various enzymes of different gene families.
These modifications are present in organelles. And since RNA metabolism occurs in these
organelles as well as in cytosol, characterizing the role that these enzymes have in mRNA
turnover is important.
“Nudix” Hydrolase is a family of widely distributed pyrophosphohydrolase enzyme found in
all classes of organisms. Catalysis depends on Nudix motif which is 23 amino acids long. The
domain structure is formed by two β-sheets packed between α-helices that is well adapted to
binding and hydrolysis of a wide range of nucleoside (McLennan 2006). The defining
characteristic of Nudix enzymes is their ability to catalyze the hydrolysis of a variety of
nucleoside diphosphate derivatives with varying degrees of specificity (Kraszewska 2008).
Nudix hydrolases could be considered “housecleaning” genes whose function is to cleanse the
cell of potentially deleterious endogenous metabolites and to modulate the accumulation of
intermediates in biochemical pathways (Bessman et al. 1996). In E. coli hydrolysis of 5’
terminal triphosphate to monophosphate is catalyzed by a member of the Nudix hydrolase
family called RppH (RNA pyrophosphohydrolase).
1.8 RNA pyrophosphohydrolase (RppH)
RNA pyrophosphohydrolase (RppH) formerly designated as NudH/YgdP is a recently
discovered protein that has RNA pyrophosphohydrolase activity in E. coli. It is a member of
the Nudix family with the Nudix signature motif GX5EX7REUXEEXGU where U is a
hydrophobic residue and X is any residue (McLennan 2006). It catalyzes the conversion of a
5’ triphosphosphate primary transcript to a 5’monophosphate RNA, assisting in the 5’→3’
mRNA degradation (Deana et al. 2008). Like in E. coli, RNA pyrophosphohydrolase in B.
subtilis that catalyzes this event is a Nudix protein that prefers unpaired 5' ends. However, in
B. subtilis, this modification exposes transcripts to rapid 5' exonucleolytic degradation by
RNase J, which is absent in E. coli but present in most bacteria lacking RNase E (Richards et
al. 2011). Yet Chlamydomonas appears to encode RNase J (Stern et al. 2010).
Recent findings that goes along with previous works show that the 5’-3’ rppH mediated
degradation can be regulated by ribosome binding close to the translation start site (Richards
et al. 2012). Some evidences suggest that rppH is involved in mRNA degradation in the
chloroplast. Recently an in vivo study done on degradation of transcripts in a specific reporter
gene constructs in Chlamydomonas cells shows that initiation of degradation starts at the 5′
end (Salvador et al. 2011). The same study has indicated that it is likely that an exposed 5′
terminal nucleotide with its phosphate group as the binding site of a protein (trans acting
factor) that initiates mRNA breakdown in chloroplasts (Salvador et al. 2011) from the 5’ end.
9
Introduction
Figure 1.3. Bacterial RNA decay model. A) The removal of pyrophosphate by rppH enhances the the
RNase E compartment binding to 5’ monophosphorylated mRNA thereby activating degradation of
mRNA in E. coli. B) RNA decay in bacteria containing the exonuclease RNase J (B. subtilis),
endonuclease activity exposes the primary transcripts to 3’ exonuclease or RNase J attack. In addition
pyrophosphate removal by rppH analogue results in degradation of monophosphorylated transcript by
5’ exonulease activity of RNase J (Belasco 2010).
1.9 Sub-cellular localization
Transcripts are thought to contain localization signals, consisting of discrete stem loop
structures found at the 5’ end of a gene which are recognized by transacting factor for the
screening of localized transcripts among the general population of RNAs .
Cellular asymmetry in Chlamydomonas is determined by the anterior and posterior position of
the flagella and chloroplast respectively with the nucleus in between these two organelles.
The nuclear architecture of Chlamydomonas is organized as a series of concentric spheroids,
the innermost being the nucleolus that serves as the site for the ribosomal RNA synthesis.
Surrounding the nucleolus is a spherical compartment that presumably contains the sites of
synthesis and processing of pre-messenger RNAs in Chlamydomonas. Nuclear organization is
linked to cytoplasmic events such as transcript targeting (Colon-Ramos et al. 2003).
1.10
GFP
Bioluminiscence in cnidaria is due to oxidation of luciferin via luciferase or Ca2+-activated
photoprotein that excites a class of proteins called green-fluorescent proteins (GFPs) (Prasher
et al. 1992). GFP, first named (Shimomura et al. 1962) as “Aequorin”, was shown to have
10
Introduction
emission of light on addition of Ca2+ ions. Unlike the other fluorescent proteins A. victoria
wild type GFP has chromophores in protonated state.
The green fluorescent protein (GFP) from the jellyfish A. victoria is a 238 amino acid protein
that exhibits bright green fluorescence under ultraviolet blue light exposure. Neutral wild type
GFP has a dual absorption wave length at 395nm and 475nm and emits green light at 509nm
(Brejc et al. 1997). Crystallographic structural analyses on the wild type and mutant S65T
GFP has shown that, this dual absorption spectra is caused by a change in the ionization state
of the chromophore, which is the structural unit of GFP.
GFP tagging permit analyses of proteins in living cells easier and offer distinct advantages
over conventional immunofluorescence. Among these are lower background, higher
resolution, and avoidance of fixation artifacts (Michaelson and Philips 2006). Various GFP
variants have been mutated to give reporters of different spectral properties so that multicolor
labeling is now a practical option (Paddock 2008). Transgenic expression of GFP within any
given cell requires simply placing the optimized versions of GFP sequence under the
transcriptional control of appropriate regulatory sequences and in the correct reading frame
(Chytilova et al. 1999). The spectral properties of the recombinant GFP suggest that
production of the fluorescence is not species-specific (Chalfie et al. 1994).
11
Aim of the project
1.11
Aim of the project
The aim of this project was to develop a reporter gene construct that could be used to localize
RNA pyrophosphohydrolase in C. reinhardtii cells. The project had also sub-aims;
 Selection of a reporter Green fluorescent protein (GFP).
 Codon optimization of Zs Green 1 from Zoanthus Sp. to codon usage in the nucleus of
C. reinhardtii.
 Linking the codon optimized GFP to the 5’rppH region which is thought to have the
localization signal for RNA pyrophosphohydrolase.
 Construction of pE coli -5’rppH-GFP vector to express GFP construct in E. coli.
 Construction of transformation vector pChlami-5’RppH-GFP that could be used to
transform C. reinhardtii.
 Nuclear transformation of C. reinhardtii.
 Selection and screening of positive C. reinhardtii transformants by PCR.
12
Materials and methods
2. Materials and methods
2.1 PCR
By using thermocycler (Biometra), standard PCR procedure was followed to amplify DNA
fragments by using Taq polymerase. Amplification of fragments for cloning experiments was
carried out in three steps, two denaturation cycles at 98˚C for 1 min and 30 sec respectively,
30 annealing cycles at 65˚C for 30 sec, and two elongation steps at 72˚C for 1 min in 30
reaction cycle. In all PCRs, the primer concentrations were 10 pMol/µL. For genomic DNA
(1 ng/ µL) amplifications, the annealing temperature were different for different reactions
involving different primers (see Appendix V). Since different primers that were used have
different melting temperatures, the annealing temperature was adjusted accordingly.
2.2 Construction of fusion proteins and vectors
Standard molecuar biology techniques were used as explained in (Sambrook and Russell
2001), and all restriction enzymes for vector digestion was done according to the
manufacturers general guide lines (New England Biolabs). Furthermore, for all restriction site
check up; for ligation or specific sequence analyses, was done by NEB cutter V 2.0 (New
Englands BioLabs). The following vectors were constructed for the expression of the fusion
protein in which the 5’-rppH region (207 ), was fused to the N-terminus of the optimized
GFP. An extra amino acid (GLY) was added inorder to have the correct reading frame of GFP
by including an extra nucleotide [C] just upstream of the start codon of GFP. The synthesized
lyophilized plasmid containing GFP gene was manipulated according to the manufacturers
instruction (Life technologies) for further use. SmaI and EagI sites were used for cloning of
GFP to a pBluescript II SK+-5’rppH vector received from Uwe Klein. GFP which is flanked
by SmaI and EagI sites was cloned into pBluescript II SK+-5’rppH at the SmaI and EagI sites
which was digested with SmaI and NotI restriction enzymes. The 5’rppH-GFP gene was then
amplified by one PCR thereby introducing the NcoI restriction site in the PCR product. See
Appendix V for primers.
The PCR product was purified by using gel purification kit (GE Healthcare), cut with
restriction enzymes Ncol/Notl and was cloned into pE coli vector (Clontech) after digesting
the vector with NcoI and NotI enzymes. The 5’rppH-GFP fragment was then amplified by 3
PCR reactions to incorporate a new NdeI site and replace the Ncol site in pE coli vector.
Three Ndel forward primers and Xbal reverse primer were used for the oligonucleotide [CAT]
synthesis (see Appendix V). At each interval of the PCR, amplified PCR products were run on
agarose gel (1%) and purified. Subsequently each purified PCR products were diluted to meet
the starting template amount required for the next PCR.
After purification of the final PCR product from a gel, the NdeI/XbaI digested PCR product
was cloned into the pBluescript II SK+ vector (Uwe Klein). The vector contains XhoI-XbaI
13
Materials and methods
gene fragment that is taken from pChlamiRNA3int vector (Molnar et al. 2009); for clarity this
vector is named as an ‘intermediate vector’ from now on.
To form the transformation vector, the XhoI-XbaI fragment which was digested from the
intermediate vector was cloned into pChlami vector (Molnar et al. 2009) which had released
its original XhoI-XbaI fragment region after restriction cutting to form the pChlami-5’rppHGFP vector. In the next sections the term pChlami vector is used to refer to the
pChlamiRNA3int vector that has released the rbcS2-intron sequence.
2.3 Agarose gel electrophoresis
Determination of the size of DNA was done by running DNA samples on a 1% agarose gel
containing ethidium bromide [0.17 µg/mL] in TAE buffer (see Appendix I). A 1Kb plus
standard ladder was used to estimate the size of the DNA fragments or PCR products. The gel
was run at 90 V for 30 min, and for longer durations when separation of smaller bands were
needed. Pictures were taken for further analyses.
2.4 Ethanol precipitation
All DNA samples were precipitated by adding 1/10 volume of Na acetate [3M] and 2×
volume of 96% ethanol on ice for 30 min. Centrifugation at 4˚ C, washing with 70% ethanol
was respectively done afterwards.
2.5 Bacterial cells
TB1 E. coli strain was the host bacteria that was used for all plasmid and vector
transformation in this study except for recombinant protein expression analyses. Procedures
for E. coli growth, transformation, maxipreps and minipreps was done by following the lab
manual and coarse book (Uwe Klein). All E. coli transformants were grown on agar plates
containing ampicillin (60 µg/mL) as a selection marker, and overnight growth of cultures was
done for all on LB medium (See Appendix I) supplemented with ampicillin (100 µg/mL).
2.6 Transformation of Chlamydomonas
Nuclear transformation of the cell wall-less Chlamydomonas strain cw-15 cells, was done by
Uwe Klein, by following the protocol of (Kindle et al. 1989). Cells were vortexed in the
presence of linearized DNA (transformation vector), glass beads and polyethylene glycol
(PEG). Cells were plated out on paromomycin containing agar plates and incubated under
sterile condition.
14
Materials and methods
2.7 Selection of transformants
Individual colonies were picked once their sizes were clearly visible on the paromomycin
(50mg/mL) containing agar plate. Each colony that was picked was marked at the back of the
plate.
2.8 Cell culture
The mutant strain cw-15 of C. reinhardtii was obtained from cell culture in Chalmaydomonas
genetic center at duke university NC, USA. Transformants of the alga grown on paromomycin
containing agar plates were inoculated in 100-mL liquid culture. Cells were maintained on HS
(high salt) (Sueoka 1960) medium supplimented with [salt stock, phosphate stock and trace
elements, see Appendix I] at ~24˚ C on light intensity ~ [500 W/m2]. Cells were supplimented
with 2% CO2 in bubbling water chamber (water- bath supplimentd with air) until the growth
confluency is about ~ 2×106 cells/mL at 32˚C in 12 hours light and 12 hours dark cycle. For
genomic DNA isolation purposes, cells were collected from the cell culture according to
laboratory manual (Uwe Klein).
2.9 Selective marker
Different marker genes are used for developing of transformation in Chlamydomonas. For this
experiment, the aphVIII gene (encodes for aminoglycoside 3'-phosphotransferase) was used
which confers paromomycin resistance to the Chlamydomonas cells. aphVIII has a codon
usage similar to Chlamydomonas genes (GC content of 68.9%), thereby expression of this
gene in Chlamydomonas cells doesn’t creat random mutations (Sizova et al. 2001). Usually
expression of a gene from chimeric gene constructs requires strong, constitutive or inducible
promoter system. Previously a linearized plasmid used to express aphVIII under the control of
RBCS-HSP70 chimeric promoter and the RBCS terminator generated ~22,000
Chlamydomonas transformants (Gonzalez-Ballester et al. 2005). Moreover high sensitivity of
C. reinhardtii cells to paromomycin and the efficient inactivation of paromomycin by
aphVIII, made the aphVIII gene a good candidate (Sizova et al. 2001). Concentration of
marker gene will impact the number of transformants obtained per transformation event and
the number of integrated marker gene copies per transformant.
2.10
Codon optimization
Codons used frequently in a certain organism are often not used in others. This causes a
change in the number of tRNAs and significantly affects the translation efficiency. GFP
(Clontech) which was derived from Zoanthus Sp. was opimized to C.reinherdtii nuclear
codon sequence (Nakamura et al. 2000). Graphical analyses of codon usage was done by
using Graphical Codon Usage Analyzer (Fuhrmann et al. 2004).
15
Materials and methods
2.11
Screening of transformants with PCR
PCR was used to check whether C.reinherdtii wild type cells have integrated the transgene.
Template DNAs from C.reinhardtii genome of selected transformants were amplified by
different primers to analyze the PCR product. The list of primers and the melting temperatures
can be reffered (Appendix V).
2.12
Recombinant protein expression in E. coli (BL21DE3)
The pE coli expression vector system (Clontech) contains the hybrid T7 lac promoter as well
as a lacI gene, which encodes Lac repressor. In lac hybrid promoter system (Clontech), basal
expression of the protein of interest is repressed by the Lac repressor (lacI), that binds to the
lac operator, preventing expression of genes from the promoter p lac in the absence of IPTG.
Isopropyl β-D-1-thiogalactopyranoside (IPTG) is a lactose metabolite in which up on
addition, causes allosteric conformational change in the repressor (lacI), allowing T7 RNA
polymerase to gain access to the promoter and initiate transcription of gene coding for betagalactosidase.
Figure 2.1. Recombinant protein expression in E. coli (BL21DE3) transformed with pE coli-Cterm 6HN vector. Induction of E. coli (BL21 DE3) cells with IPTG enables the T7 RNA polymerase
machinery to initiate transcription and translation. Basal protein expression is inhibited by lacI in the
absence of IPTG (Clontech).
The vector also contains an ampicillin resistance gene (Ampr) and a pBR322 origin of
replication, which maintains the vector at a low-copy-number to reduce basal levels of the
protein of interest. An origin of replication is sequence of DNA at which replication is
initiated in plasmids. It determines the vector copy number. The pBR322 replication origin is
a site from where two RNAs (RNA I and II) are transcribed. RNA I serves as the primer
initiating transcription which is attached to the 5’ RNA II sequences which is stabilized by
rop gene product. Hence the stabilization prevents change in the conformation of RNA II
which would otherwise lead to RNAse H cleavage. Therefor this stabilized bond between the
16
Materials and methods
two RNAs maintain replication initiation from a single site or origin and inturn the
mechanism should also maintain the number of copies of the vector at low level.
E. coli strain (BL21DE3) was transformed with pE coli-5’rppH-GFP vector. The
transformants were grown on agar plates containing ampicillin at 37˚C. A 3 mL LB/Amp
culture was inoculated and was grown overnight. The next day, 2 mL of the overnight culture
was inoculated in 100mL fresh LB/Amp medium. The culture was incubated on a shaker (200
rpm) for 2 hours (until the OD reaches 0.6-0.8). The cell culture was induced by adding IPTG
to a final concentration of [0.22 ng/µL]. Cells were continously induced for 6 hours on a
shaker (200 rpm). Samples were centrifuged at 5000 rpm/5 min, supernatant was descarded
and pellet was resuspended in buffer A (Appendix II).
2.12.1 Purification of Histidine-tagged GFP
BL21DE3 cells expressing His-tagged GFP (always kept on ice) suspended in low imidazole
buffer A (Binding/wash buffer, see Appendix II) were thawed, sonicated three times 5/10 sec
burst on /off cycle while maintaining the samples on ice, then centrifuged at 4˚C for 10 min,
at 10,000 rpm. Sequential extraction of samples and washing was done according to the
laboratory manual (Uwe Klein).
Preparation of TALON Co resins (Clontech), was done according to the manufacturers guide
lines. 200 μL TALON Co resin was centrifuged at 1500 rpm for 5 min. Supernatant was
discarded and the resin was resuspended in 1.5 mL dH2O and centrifuged once more at 1500
rpm for 5 min. The procedure was repeated with buffer A instead of dH2O. The resin was then
added to Empty PD-10 Desalting column (Biorad).
2.13
SDS PAGE
In sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), proteins are
separated according to their molecular mass under denaturing conditions. Treatment with the
anionic detergent SDS in heating environment (92˚C) destabilizes secondary and tertiary
structures, thus denatured proteins gain a uniform net negative charge thereby movement of
proteins on the gel depends on their size.
A 12% polyacrylamide solution was prepared by using solutions; dH2O, A, B and APS
(Appendix II) by carefully mixing. The gel was poured into a casting strand between glass
plates leaving 1 cm of space between the top of the short plate and the resolving gel level. The
top part was filled with water. After 30 min polymerization, the floating water was removed
from the top. Stacking solution was prepared by carefully mixing solutions dH2O, A, B’, and
APS (Appendix II). The stacking solution (4%) was quickly added to the top of the 12%
polyacrylamide solution. Then the comb was inserted on top before the stacking solution
polymerizes. After 30 min polymerization, the comb was taken out and the space between the
glass plates was rinsed with water to get rid of bubbling. The electrophoresis was done at 90
V for 15 min and susequently at 120 V for 30 min.
17
Results
3. Results
3.1 Optimization of GFP
Sequencing of C. reinhardtii nuclear genome revealed a high GC content (65%). Usage of
codons specific for Chlamydomonas nuclear genes is essential. Previous works have shown
that adaptation of codon usage of transgenes greatly enhances the expression of transgenes
because of differences in tRNA abundance (Leon-Banares et al. 2004). It significantly
improves translation and even promotes efficient integration into the genome (MesletCladiere and Vallon 2011).
This study focused mainly on development of a reporter construct for the localization of the
putative RNA pyrophosphohydrolase in C. reinhardtii. We used ZsGreen 1 GFP as reporter
molecule from (Clontech), which has a size of 699 bp because of its bright green fluorescence
with excitation, and emission maxima at 493 and 505 nm, respectively. GFP was codon
optimized using the Codon Usage Database (Nakamura et al. 2000). Standard format was
used for code selection. The database lists C. reinhardtii’s codon usage in genes and sum of
codon; used. The codon usage table lists the relative frequency of each codon for a particular
amino acid. Triplet codons are grouped according to the fraction of each triplet in the total
nuclear genomic sequences and the frequency of each triplet per thousand bp of genomic
sequences (see Appendix IV).
By using the GCUA tool (Fuhrmann et al. 2004), the GFP sequence was split into triplet , and
the frequency of each codon was compared to that of 420,455 nuclear C. reinhardtii codons
from the codon usage table. Thus, the relative adaptiveness defined by the GCUA tool shows
the percentage of each codon as compared to that of the most preferred C. reinhardtii codon.
Each GFP codon was manually optimized to codons that are most preferred and frequently
used in the total nuclear C. reinhardtii genome. Almost all codons of GFP were optimized to
the most preferred C. reinhardtii nuclear codons as shown in fig. 3.1.
The sequence was further modified prior to GFP synthesis for cloning into the pE coli-Cterm
6xHN (Clontech) vector. This vector encodes a 6xHN tag composed of 6 repeating His-Asn
subunits. For the purpose of GFP expression and purification in E. coli cells, the 6xHN tag is
incorporated in our gene construct. The stop codon for GFP was removed and an extra [C]
nucleotide was included just upstream to the start codon of GFP. The optimized and adjusted
GFP sequence was then synthesized (Life technologies).
18
Results
Figure 3.1. Graphical Codon Usage Analyses. GFP (Zsgreen1) has 699 total codons. Most codons
were optimized to a relative adaptiveness (vertical line) of > 75% (black bar). Codons that have
relative adaptiveness of < 20% are also shown (grey bar). The analyses was done by (GCUA)
(Fuhrmann et al. 2004).
19
Results
3.2 Cloning
Step by step cloning of the synthesized GFP is shown in fig. 3.2. There are four cloning steps
designated as A, B, C and D.
20
Results
Figure 3.2. Cloning of GFP. Four steps (A-D) of cloning procedures and the restriction sites used are
shown. Step A; GFP was cloned into pBluescript-5’rppH vector. Step B; 5’rppH-GFP was cloned into
pE coli-C-term vector. Step C; 5’rppH-GFP was cloned into intermediate vector. Step D; 5’PSAD5’rppH-GFP was cloned into pChlami vector (transformation vector). Red color-5’PSAD promoter
sequences; Grey color-5’rppH region; Black color-GFP; Yellow color; 3’PSAD terminator sequences.
3.2.1 pBluescript II SK+ 5’ rppH-GFP vector: Cloning step A
For the purpose of localization of RppH in C. reinhardtii, the 5’ region of RppH was tagged
to GFP to be able to follow the localization of the GFP-tagged gene product. Although not
proven, a signal peptide that directs localization of the RppH protein was assumed to be
present in the 5’ region of RppH.
The pBluescript II SK+-5’rppH vector was received from Uwe Klein. It is a pBluescript II
SK+ with 2961 bp (stratagene) that has the 5’ region of RppH. GFP was cloned into
pBluescript II SK+-5’rppH at the Smal and Eagl sites to form pBluescript II SK+-5’rppHGFP as shown in fig. 3.2 A and fig. 3.3. The restriction site for Smal (CCCGGG) is found one
nucleotide upstream of the start codon of GFP i.e. at the end of the 5’rppH sequence, and
Eagl (CGGCCG) is found at the end of the coding region of GFP (see Appendix III). By
restriction cutting, the vector fig. 3.4 (pSK+-5’rpph-GFP), at EcoRV/Eagl restriction sites
and subsequent gel-electrophoresis analyses, the vector released a fragment which is ~ 953 bp
in size as expected (fig. 3.4). The vector used for cloning pBluescript II SK+-5’rppH fig. 3.4
21
Results
(C) was also cut with the same restriction enzymes. From the gel electrophoresis analyses, it
can be seen that the restriction digested vector released a band (lower) of ~ 250 bp
corresponding to the 5’ rppH region (see fig. 3.4 ).
Figure 3.3. Restriction map for pBluescript II SK+-5’rppH-GFP. The 5’ rppH is represented by vertical
lines. GFP is represented with diagonal lines. The different restriction sites present in 5’ rppH-GFP gene
fragment (SmaI, EagI) as well as in the pBluescript SK+ vector (EcoRV, PstI) are shown. EcoRV site is found
253 bp upstream to the start codon of GFP in the pBluescript II SK+ vector. SmaI and EagI flanked GFP region
has a size of 700 bp. Dotted line represents sequences in the pBluescript II SK+ vector.
Figure 3.4. Gel electrophoresis analyses of pBluescriptII SK+-5’rppH-GFP vector cut with
EcoRV/Eagl. Lane 1 (L); is 1kb+ ladder. Lane 2 (pSK+-5’rppH-GFP); is restriction digested
pBluescriptII SK+-5’rppH-GFP construct. Lane 3 (C); is pBluescript II SK+-5’rppH used for cloning
as a control.
22
Results
3.2.2 pE coli-C-term-5’rppH-GFP 6×HN vector: Cloning step B
The 5’rppH-GFP gene was amplified by PCR thereby introducing an NcoI restriction site in
the PCR product. After cutting the PCR product with Ncol/Notl restriction enzymes, the
5’rppH-GFP gene flanked by Ncol/Notl sites was cloned into Ncol/Notl restriction sites of pE
coli-C-term 6×HN vector with the same enzymes as shown in fig. 3.2B. The ligation was
checked with restriction digest of the ligated vector (pE coli-5’rppH-GFP-6HN) by using
Ncol/Xbal, that releases a weak fragment which is ~ 990 bp as shown in fig. 3.6.
In the control experiment fig. 3.6 (C), the restriction digest releases a fragment that
corresponds to the multiple cloning sites (MCS) in the pE coli-C-term 6×HN vector
(Clontech) between NcoI/XbaI sites. But since the size is very small and the band is too weak,
it is not seen in the gel electrophoresis analyses.
Figure 3.5. Restriction map of 5’rppH-GFP in pE coli vector. The map shows the different
restriction sites NcoI, SmaI, NotI, XbaI. +1 refers to the translation start site of the fusion protein
[5’rppH-GFP-6×HN (boxed)] until the XbaI site. Single dot line represent 39 nucleotide sequences
that are present in the pE coli-C-term 6×HN vector (MCS). The vertical lines represent 5’rppH (207
bp) and the diagonal lines represent the GFP region.
23
Results
Figure 3.6. Gel-electrophoresis analyses of pE coli 5’rppH-GFP vector digested with Ncol/Xbal.
Lane 1 (L) is 1 kb+ ladder; Lane 2 (VC) and Lane 3 (C); are restriction digested vector construct and
pE coli C-term 6×HN vectors (used for cloning) respectively with Ncol/Xbal.
3.2.3 Intermediate 5’ rppH-GFP-6×HN vector: Cloning step C
The intermediate vector which was received from Uwe Klein, is a pBluescriptII-SK+ vector
that has a 1233 bp Xhol/Xbal fragment taken from the pChlamiRNA3int vector (Molnar et al.
2009). This fragment has a Chlamydomonas 5’-PSAD promoter region from the XhoI site
until the NdeI site as can be seen in fig. 3.8.
Figure 3.7. pChlamiRNA3int vector background. Vector map showing the restriction sites,
selective marker (aphVIII) and the regulatory promoter and terminator regions (Molnar et al. 2009).
24
Results
One of the characterstic features of nuclear genes is the presence of introns in the coding
region. Transgene expression was shown to significantly increase by the presence of an intron
in constructs used for ectopic expression in Chlamydomonas (Lumbreras et al. 1998). The
RBCS2 intron has been widely used in transgene expression in C. reinhardtii. Although this
system is possible, it takes time to insert the RbcS2 intron into the coding sequence. The
nuclear encoded PsaD protein which has a size of 20 kDa, encodes an abundant chloroplast
protein found in the stromal side of the photosystem I complex. The psaD reading frame
doesn’t have any introns implying that the regulatory sequences should reside in the 5’ and 3’
untranslated regions.
For cloning of 5’rppH-GFP gene into the intermediate vector, a restriction site (Ndel) was
introduced by PCR at the translation start site of pE coli 5’rppH-GFP (see Appendix V). The
Ndel site was introduced in 3 PCRs (section 2.2). Each PCR amplification gave a product that
changed a single nucleotide. Three different 5’ primers and a single 3’ primer at the Xbal site
were used for oligonucleotide synthesis. The PCR product which is flanked by the Ndel site at
5’ end and Xbal site at the 3’ end was then cloned into the Ndel/Xbal digested intermediate
vector. Cloning of the PCR product was checked by digesting the vector with Pvull which
cuts GFP at two sites that are 186 bp apart (see fig. 3.8) and at two other sites in the vector
background (see fig 3.9). The restriction digest was run on a 1% agarose gel (fig. 3.9).
Figure 3.8. Restriction map of 5’PSAD-5’rppH-GFP-6×HN gene segment in the intermediate
vector. Shown here are restriction sites (XhoI, NdeI, SmaI, PvuII, NotI and XbaI) relative to +1 which
is the translation start codon. The 5’ PSAD promoter region is shown in black, vertical and diagonal
lines represent 5’ rppH and GFP regions respectively. Bold dots represent sequences that are part of
the vector.
25
Results
Figure 3.9. Gel electrophoresis analyses of PvuII digested Intermediate vector. Lane 1 (L); is
1kb+ ladder, Lane 2 (I); is an intermediate vector with gene construct (5’rppH-GFP) and Lane 3 (C);
is an intermediate vector without the gene construct (5’rppH-GFP) used as a control.
3.2.4 Transformation vector pChlami-5’rppH-GFP-6×HN: Cloning step D
Finally, the gene fragment flanked by Xhol/Xbal was cloned into pChlami vector. First
pChlamiRNA3int vector was digested by cutting the vector at Xhol/Xbal sites. The gene
fragment flanked by Xhol/Xbal was also cut out from the intermediate vector and ligated into
the opened transformation vector at the Xhol/Xbal sites. It is important to note here that the
transformation vector has no longer the rbcS2-intron sequence which is present in the original
pChlamiRNA3int vector (fig 3.7).
Figure 3.10. Restriction map of 5’PSAD-5’rppH-GFP-6×HN in pChlami vector. Shown here are
positions of restriction sites XhoI, NdeI, SmaI, XbaI and SmaI relative to +1 which is the translation
start codon. The 5’ PSAD promoter region is shown in black; 5’ rppH and GFP regions are
represented in vertical and diagonal lines respectively. The 3’ PSAD terminator sequence is shown in
dotted gray color.
Cloning of the insert in the transformation vector was checked by restriction cutting the vector
at Xhol/Xbal site and subsequent agarose gel electrophoresis analyses. The expected insert
size from the Xhol site to the Ndel site and further to the Xbal site is 1814 bp see fig. 3.10.
26
Results
This fragment (approximate size) was released when analyzed by gel electrophoresis as can
be seen in fig.3.11.
Figure 3.11. Gel electrophoresis analyses of pChlami- 5’rppH-GFP-6×HN vector digested with
Xhol/Xbal restriction enzymes. Lane 1(L) is 1kb+ ladder; Lane 2 (pC) is digested pChlami- 5’rppHGFP-6×HN vector releasing a fragment of ~ 1810 bp (lower band); Lane 3 (C) is digested
pChlamiRNA3int vector (control) used for cloning releasing a fragment of ~ 1300 bp (lower band).
The difference between the two lower bands corresponds to the difference between the two (pC and C)
total vector sequences (581 bp).
3.3 Transformation and selection of transformants
The cell wall-less Chlamydomonas strain cw-15 cells were transformed by Uwe Klein as
explained in section 2.6. Cells were grown on agar plates containing paromomycin (section
2.7). Cells which have integrated the marker gene (aphVIII) were selected. It is impossible to
conclude that cells that have exhibited resistance to paromomycin are all positive
transformants for the GFP construct. This is because of random integration of sequences into
the Chlamydomonas nuclear genome. Thus further screening for GFP positive transformants
at the genomic DNA level was needed.
27
Results
3.4 Analyses of transformants by PCR
Genomic DNA was isolated as described in section 2.8. Identification of cells harboring the
introduced gene at the genomic level was done by PCR. More than 15 transformants that were
resistant to paromomycin were analyzed. And we proceeded with five transformants that we
thought were GFP positive. A combination of different primers was used to amplify specific
sequences of genomic DNA at different sites as can be seen in fig.3.12.
Figure 3.12. Map of the transformation vector (pChlami 5’PSAD-5’rppH-GFP-6×HN) with the
different primers that were used to amplify specific sequences by PCR. Each primer (boxed) and
the respective starting nucleotide positions in each strand depicted are shown relative to +1 which is
the translation start codon. The 5’ PSAD promoter region is shown in black; 5’ rppH and GFP regions
are represented in vertical and diagonal lines respectively; 6×HN until the XbaI site (see fig. 3.5) is
shown as a background color. The 3’ PSAD terminator sequence is shown in dotted gray color. The
selective marker gene (aphVIII) is shown in gray. Dotted lines represent sequences in the pChlami
vector.
28
Results
Genomic DNA from the five transformants were amplified by 5’ PSAD6401 and 3’ PSAD495 (see
fig. 3.13). As a positive control, template gene fragment from the tranformation vector was
amplified by the same primers. Similarly, as a negative control, Chlamydomonas wild type
genome was used as a template.
Figure 3.13. Gel electrophoresis analyses of PCR products using genomic DNA as a template.
Lanes 1-5; represent transformant’s (samples 1-5 respectively) genomic DNA amplifications. The
PCR gave a product which has a size of ~ 780 bp that corresponds to the endogenous psaD. Lane 6
(+Ve) positive control; is amplified template gene fragment from the transformation vector; Lane 7
(WT); is the negative control (amplified wild type, Chlamydomonas genomic DNA as a template). The
positive control (+Ve) shows amplification at ~ 780 bp (lower band) and at 1178 bp (upper band). The
latter is the expected band size from this PCR. The negative control also shows amplification as
expected at ~780 bp (psaD) gene size. Lane 8 (L); is ladder used (1Kb+).
The above figure shows that the genomic DNA of the five transformants have a PCR product
which is ~ 780 bp that corresponds to a fragment from the endogenous psaD gene. The
expected PCR product ~ 1178 bp, in the positive control (amplified template from
transformation vector), upper band is also shown in fig. 3.13. On the negative control, the
primers have amplified a gene fragment that has a similar size to the PCR products from the
genomic DNA of the transformants and to the positive control (lower band). In the negative
control (amplified wild type, Chlamydomonas genomic DNA as a template), the expected
PCR product is 778 bp that corresponds to the wild type endogenous psaD gene from the
primers used for this amplification.
29
Results
Next, a specific genomic DNA fragment was amplified by using NUD3’-5COMP and 3’GFP
primers (see fig. 3.14). These primers amplify a gene fragment that has a size of 205 bp (see
fig. 3.12). The amplification reaction gives PCR products that are 205 bp in size. This is
expected from this PCR reaction. The positive (amplified template from transformation
vector) and negative controls (amplified wild type, Chlamydomonas genomic DNA as a
template) were also included in the PCR. The positive control has a PCR product that is
similar in size to that of the transformants (samples 1-5 respectively) genomic DNA
amplification. In the negative control, the DNA amplification has a size of ~ 650 bp. This is
unexpected in a way that one of the primers (3’GFP) is not supposed to amplify the wild type
genomic DNA used as a template, since GFP is not present in the Chlamydomonas genomic
DNA.
Figure 3.14. Gel electrophoresis analyses of PCR products using genomic DNA as a template.
Lane 1(L); is 1kb+ ladder used. Lane 2-6 (sample 1-5 respectively); represent transformant’s genomic
DNA amplifications. Lane 7 (+Ve); is amplified transformation vector Lane 8- (WT); is the negative
control (amplified wild type, Chlamydomonas genomic DNA as a template). Samples 1-5 have PCR
products at 205 bp which is similar to the positive control. Amplification product in the negative
control has a size of ~ 650 bp.
The transformants were also screened for the gene that confers paromomycin resistance which
is present in the transformation vector but absent in the wild type genomic DNA. Figure 3.15
shows PCR products of gene fragments amplified by using 5’paro4331 and 3’paro4743 primers
(see fig 3.15). The PCR products from all samples (1-5 respectively) have size of 433 bp. The
positive control (amplified template from transformation vector) also has a PCR product of
approximately the same size. The wild type Chlamydomonas lacks the gene that confers
30
Results
paromomycin resistance, aphVIII. Thus, no amplification is seen in the negative control as
expected.
Figure 3.15. Gel electrophoresis analyses of PCR products using genomic DNA as a template.
Lanes 1-5 (samples 1-5 respectively); show PCR amplification at ~ 430 bp as expected. Lane 6 (+Ve);
is transformation vector that shows amplified fragment, which has a size of ~ 430 bp. Lane 7 (WT); is
negative control (amplified wild type, Chlamydomonas genomic DNA as a template) which shows no
amplification since it lacks the aphVIII. Lane 8 (L); is 1Kb+ ladder.
Finally transformants (samples 1-5 respectively) were screened by using 5’NUD GFP and 3’
PSAD 495 (see fig. 3.16). From the gel electrophoresis result, only transformant number 5
(sample 5) gives a PCR product that has a size of ~1070 bp as expected. The amplified gene
fragment of the positive control (transformation vector) also has the same size (1070 bp). The
negative control (amplified wild type, Chlamydomonas genomic DNA as a template) shows
weak products that have sizes of ~ 200 and another at ~ 1000 bp.
31
Results
Figure 3.16. Gel electrophoresis analyses of PCR products using genomic DNA as a template.
Lanes 1-5 (samples 1-5 respectively). Lane 5 (transformant number 5); shows a PCR product that has
a size of ~ 1070 bp. Lane 6 (+Ve); is amplified transformation vector that gives a PCR product which
has a size of ~ 1070 bp similar to PCR product from transformant number 5. Lane 7 (WT); is a
negative control (amplified wild type, Chlamydomonas genomic DNA as a template) that shows weak
bands at ~200 bp and at ~ 1000 bp. Lane 8 (L); is 1Kb+ ladder.
3.5 Recombinant protein expression and purification
Recombinant expression of GFP from the jellyfish A. victoria was first accomplished in E.
coli (Chalfie et al. 1994). To determine if the GFP construct transformed into
Chlamydomonas is capable of producing functional GFP protein (fluorescence) in vivo, the
construct was expressed in E. coli cells as described in section 2.12. But fluorescence
microscopy was not done due to shortage of time. Rather E. coli BL21DE3 lysates were
purified by His-tag affinity chromatography using cobalt-sepharose. We examined E. coli
BL21DE3 cell lysates for the expression of the GFP fusion protein.
Figure 3.17. SDS-PAGE analyses of E. coli BL21DE3 lysates purified by Co sepharose affinity
chromatography. Lane 1; is prestained protein ladder. Lane 2 (CE); is crude extract showing all
proteins that are present in E. coli cells. Lane 3 (FT); is the flow through, all proteins that are present
in the crude extract except for the HN tagged GFP protein (5’rppH-GFP). Lane 4 (S); is the purified
cell lysate sample (transformed with pE coli-5’rppH-GFP-6×HN) that was made in this experiment.
Lane 5 (C); is a purified 5’rppH-GFP-6×HN protein as a control. The purified sample (S) does not
show any proteins that are purified. The purified sample (C) shows the expected 5’rppH-GFP-6×HN
tagged protein size which is 36 kDa.
32
Results
The SDS-PAGE analyses shows E. coli BL21DE3 lysates purified by affinity
chromatography. Crude extracts (CE) in Lane 2 in fig. 3.17, are all proteins in the cell. The
flow throw (FT), are polypeptides that have no affinity to the metal (cobalt) therefore directly
pass through the column. Due to the high affinity of histidine which is tagged to the Nterminal region of GFP, to the metal ion, the tagged protein remains bound to the resin. The
tagged protein is eluted with elution buffer which has a higher imidazole concentration than
the washing buffer. The size of the fusion protein (5’rppH-GFP-6×HN) is 36 kDa. We have
tried to express this protein in E. coli. From fig. 3.17, the construct that was made in this
experiment (S) didn’t show any purified protein signal. But other already induced E. coli cells
received from Uwe Klein represented as (C) on fig. 3.17 shows a band that is ~ 36 kDa in size
which is the expected band size for the fusion GFP protein.
33
Discussion
4. Discussion
To develop a reporter gene for expression in the C. reinhardtii nucleus, a codon optimized
GFP gene was synthesized. The reporter GFP fused to the 5’ region of the rppH gene was
used to transform C. reinhardtii cells. Transformants were then screened at the genomic level
for GFP positives.
Codon optimization of synthetic GFP
Nuclear-based ectopic expression in C. reinhardtii is highly codon biased with G or C
preferred at the third position. Events causing gene silencing in C. reinhardtii are related to
inappropriate nucleotide sequences (Fuhrmann et al. 1999). In addition, previous reports have
shown that codon usage affects the level of expression of recombinant proteins in the C.
reinhardtii (Franklin et al. 2002). Other experiments have also shown that the cellular tRNA
abundance is correlated with the number of tRNA genes and is adjusted to the codon usage to
optimize translation efficiency in C. reinhardtii (Cognat et al. 2008). Moreover the extent of
codon bias for each gene is related to the protein production level (Nakamura et al. 1999).
Previously synthetic GFP tagged to a protein reflecting the C. reinhardtii nuclear codon usage
was expressed in C. reinhardtii which allowed the visualization of the recombinant protein.
We have optimized GFP according to the nuclear C. reinhardtii codon usage preference by
using Codon Usage Database. The fraction of each codon usage from the GFP was computed
to that of C. reinhardtii nuclear genome codon usage. Among the total 233 codons in GFP,
198 were optimized to the most frequently used codons in C. reinhardtii i.e. 100% relative
adaptiveness as defined by the GCUA tool. 4 of the GFP codons were optimized to those that
are not as most frequently used, but moderately used in the C. reinhardtii nuclear genome.
There is one codon in the GFP sequence that codes for glycine (GGT) which is not optimized
at all (relative adaptiveness ˂20%). Genes encoding highly expressed proteins tend to utilize
codons whose levels of tRNAs are particularly abundant. rbcL which is highly expressed in
the C. reinhardtii genome was computed in the same way as was done for GFP. From the
analyses, it is evident that around 7% of the total codon sequence have relative adaptiveness
(as defined by GCUA) below 20%.
Expression of GFP in E. coli BL21DE3
GFP was expressed in E. coli cells to validate its function (fluorescence) in vivo. Due to
shortage of time, fluorescence couldn’t be analyzed. But E. coli BL21DE3 cell lysates were
purified by Co sepharose affinity chromatography to detect the chimeric GFP protein. SDSPAGE analyses of the synthesized GFP expressed in E. coli cells shows that the expression
level of the protein is low as seen in the control experiment. This is expected in a way that the
GFP codon sequences were not optimized to that of bacterial cells genome codon usage rather
to C. reinhardtii nuclear genome.
34
Discussion
Nuclear transformation of C. reinhardtii
Nuclear transformation in C. reinhardtii is random. Although billions of cells were used to
transform C. reinhardtii, we could only proceed with 5 transformants that integrated the
paromomycin resistance gene. We primarily used PCR for transformant screening at the
genomic level. Since results from PCR do not differentiate between genomic and ectopic
fragment amplification, we used our construct (transformation vector) as a positive control
and wild type C. reinhardtii genomic DNA as a negative control to determine positive
transformants.
Transformants were first screened for gene fragment that is amplified by primers that anneal
at the 5’ and 3’ PSAD regulatory regions. The expected PCR product amplification for the
transgene is 1178 bp. This could not be verified from this PCR for all the transformants. In
addition the wild type C. reinhardtii genomic DNA has psaD gene flanked by 5’ and 3’ UTR.
Amplification product of the negative control by the primers used gives a PCR product which
is ~ 780 bp, expected product for the endogenous amplification. In the same experiment the
positive control has amplification products of two sizes at ~ 1180 and at ~ 780 bp. This could
either be contamination with the wild type genomic DNA or that the PCR was not optimal to
amplify a template gene fragment that has a larger size. The possibility that the positive
control is contaminated is less likely that all the PCR reagents were prepared in master mix
first and that the template DNA was added to the respective reaction tubes. In addition the fact
that only the endogenous genomic DNA amplification from the transformants imply that the
PCR was not optimal (concentration, annealing temperature and elongation time) for larger
size template DNA. The PCR was optimized by altering the annealing temperature, but valid
results could not be obtained. Additional experiment can be done to optimize the PCR more
by altering the primer concentrations and adjust the concentration of magnesium ions.
We further screened the transformants with primers that amplify a gene fragment from the 5’
rppH region to the 5’ end of GFP. The PCR product for all genomic DNA from the
transformants and the positive control (transformation vector) has a size of 205 bp as
expected. The negative control (wild type genomic DNA) on the other hand has a PCR
product which was not expected in this PCR since there is no GFP in the wild type genome.
But other results (not shown) have implicated that the NUD 3’-5 COMP primer binds nonspecifically. Transformants were also screened for aphVIII gene which encodes for
aminoglycoside 3'-phosphotransferase that confers resistance to paromomycin. All the
specific transformant genomic DNA amplification with primers that amplify the aphVIII gene
sequence have product sizes of ~ 430 bp similar to the amplification product from the
transformation vector (positive control). The wild type genomic DNA lacks aphVIII therefore
no amplification is seen in the negative control.
Finally all transformants were screened with primers that amplifies a gene fragment that has a
size of 1072 bp. We found one transformant (№ 5) that is positive for this screening that has a
similar amplification product with the positive control. The wild type genomic DNA
amplification shows that there is non-specific binding of the primers. Further optimization of
35
Discussion
the primers can be applied by altering the annealing temperatures and changing the
concentration of the magnesium ions in the PCR.
36
Conclusion
5. Conclusion
Vector constructs made for this experiment were analyzed by restriction cutting analyses. All
analyses show that the vectors have incorporated the 5’ rppH-GFP gene fragment. The
transformation vector, which is the reporter vector construct was as well validated by
restriction cutting analyses.
The screening of transformants for positives has led us to choose one transformant (№ 5) that
seems to harbor the GFP transgene. Further analyses needs to be done to verify expression of
the transgene at the mRNA and protein levels. From these experiments it is possible to
conclude that transformants that have incorporated the paromomycin resistance gene don’t
necessarily have the GFP transgene.
Further work
Even though there was an attempt to express GFP by the pE coli vector construct which was
made in this study, it was not possible to detect GFP when the purified sample was run on
SDS-PAGE. Normally E. coli cells transformed with a protein expression vector induced with
IPTG (depending on the concentration, timing and length of induction) should be able to
express the protein of interest. Even though the cells were induced by following the protocol
and later with slight modification, such as various timing of induction (result not shown), it
was impossible to detect the fusion protein in all cases. Moreover, the construct has been
validated by restriction cutting as shown in fig. 3.6. Nevertheless the vector was not
sequenced. So further work can be done to sequence the vector construct and also detect the
fluorescence of the protein in E. coli cells.
In addition in the control experiment, the purified sample shows other purified proteins in
addition to what is expected for the fusion GFP protein. Thus to be certain that GFP is indeed
expressed in these E. coli cells and that the result is not an artifact of certain experimental
error, the sample could be analyzed by mass spectrometry (MS). By applying prior washing
and elution conditions, the presence of imidazole and high salt concentration can be adjusted
unless and otherwise would increase mass spectrometry backgrounds. Furthermore by using
specific antibody against GFP or HN tag, immunoprecipitation technique can be applied to
precipitate the fusion protein.
Conventional PCR provides limited information on the number of inserts (copy number). In
addition false negatives can result from problems with long-range PCR or low amounts of
DNA. Therefore further validation of the transformant can be done to have further
information on the copy number of the transgene before proceeding with the transcription
analyses. Even though Southern analyses is laborious, time consuming and requires large
amounts of high-quality DNA when compared to conventional PCR, screening with Southern
37
Conclusion
blot analyses identifies targeted transformants and permits analyses of the copy number of the
transgene.
Furthermore, before concluding that transformant (№ 5) could be used for the localization of
RNA pyrophosphohydrolase in C. reinhardtii cells, it is advisable to confirm the transcription
of the transgene. RNA can be isolated and further hybridization based experiments (e.g
Northern blot) can be done by designing specific probes. Alternatively, the RNA template can
be reversibly transcribed to synthesize cDNA, and gene expression could be analyzed by
using real-time reverse transcription polymerase chain reaction (RT-PCR) which uses
fluorescent reporter molecules to monitor the production of amplification products during
each cycle of the PCR reaction.
38
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I
Appendixes
APPENDIX I
Agarose gel electrophoresis:
TAE (50×)/L
Loading buffer
242g Tris base
0.25% bromphenol blue
57.1 mL glacial acetic acid…………0.25% xylene cyanol FF
100 mL 0.5M EDTA (pH 8.0)
LB medium/L:
30% glycerol
LBAmp/L Plates:
10 g tryptone
10 g tryptone
5 g yeast extract
5 g yeast extract
10 g NaCl
10 g NaCl
15 g agar
100 µg Amp
psaD gene
HS media
Salt stock (50×)/0.5 L:
Phosphate stock (50×)/0.5 L:
12.5g NH4Cl
47g k2HPO4.3H2O
0.50g MgSO4.7H2O
18g KH2PO4
Hutner trace elements/L:
compound
amount
water
EDTA disodium salt
50 g
250 mL
ZnSO4 . 7 H2O
22 g
100 mL
H3BO3
11.4 g
200 mL
MnCl2 . 4 H2O
5.06 g
50 mL
CoCl2. 6 H2O
1.61 g
50 mL
CuSO4 . 5 H2O
1.57 g
50 mL
(NH4)6Mo7O24. 4 H2O
1.10 g
50 mL
FeSO4. 7 H2O
4.99 g
50 mL
0.25g CaCl2.2H2O
I
Appendixes
APPENDIX II
SDS page buffers:
Solution A (Acrylamide)-[30%]
Solution B (Tris-HCl)-[1.5M]
Solution B’ (Tris-HCL)- [0.5M]
12% polyacryl amide gel:
dH2O-1.425 mL
Solution A -1.65 mL
Solution B-1.03mL
Ammonium persulfate [10%]-16.25 µL
4% Stacking gel:
dH2O-1.8 mL
Solution A-0.4 mL
Solution B’-0.75 mL
Ammonium persulfate [10%]-30 µL
1×electrode buffer:
Tris base 3 g
Glycine
14.6 g
SDS
1g
Affinity chromatography buffers:
Buffer A (Binding/wash buffer):
Sodium phosphate-[20 mM]
NaCl-[500 mM]
Imidazole, pH 7.3-[20 mM]
Buffer B (Elution buffer):
Sodium phosphate-[20 mM]
NaCl-[500 mM]
Imidazole, pH 7.3-[500 mM]
II
Appendixes
APPENDIX III
pChlami-5’rppH-GFP 6HN transformation vector sequence
1
22
82
142
202
262
322
382
442
502
562
622
682
742
802
862
922
982
1042
1102
1162
1222
1282
1342
1402
1462
1522
1582
1642
1702
1762
1822
1882
1942
2002
2062
2122
2182
2242
2302
2362
2422
2482
2542
2602
2662
2722
2782
2842
2902
2962
3022
3082
3142
CCCGCTGCCG
GACCCACTGT
CCTGTGCGGG
TTCCCGGGCA
ATGGAGGGCT
TTCAAGGGCA
GAGGACATCC
GACATCGTGG
CTGTTCGAGG
AACTGCATGT
ATGAAGAAGA
CAGGGCATCC
CGCTGCCAGT
CACTTCATCC
CACCTGACCG
CGTGGCTCTC
TAATTAATTA
tgccgggatg
ggggctaggc
gccacaggag
gcggacgtgc
attgagaaga
caggccatgt
gccgtgggat
ttaggacagc
agggggagac
tatagtgagt
aaccctggcg
aatagcgaag
tgggacgcgc
accgctacac
gccacgttcg
tttagtgctt
gggccatcgc
agtggactct
ttataaggga
tttaacgcga
atgtgcgcgg
tgagacaata
aacatttccg
acccagaaac
acatcgaact
ttccaatgat
ccgggcaaga
caccagtcac
ccataaccat
aggagctaac
aaccggagct
tggcaacaac
aattaataga
cggctggctg
ttgcagcact
gtcaggcaac
CCCCTGGTGG
GGGACCACGA
TGCTGCTGGC
TGGCCCAGAG
GCGTGGACGG
AGCAGGCCAT
TGAGCGCCGC
ACTACTTCAA
ACGGCGCCGT
ACCACGAGAG
TGACCGACAA
TGAAGGGCGA
TCGACACCGT
AGCACAAGCT
AGCACGCGAT
CGGGCGCTGC
ATCTAGAtgg
gccgatttcg
gaagggcagt
gatcaggggg
tgttgtagat
ccaaccaact
gagagtttgc
gatgcacgca
tgtgggtcag
agcgtgactg
cgtattacgc
ttacccaact
aggcccgcac
cctgtagcgg
ttgccagcgc
ccggctttcc
tacggcacct
cctgatagac
tgttccaaac
ttttgccgat
attttaacaa
aacccctatt
accctgataa
tgtcgccctt
gctggtgaaa
ggatctcaac
gagcactttt
gcaactcggt
agaaaagcat
gagtgataac
cgcttttttg
gaatgaagcc
gttgcgcaaa
ctggatggag
gtttattgct
ggggccagat
tatggatgaa
CGTTCGGCTG
GACGGGCGAG
TCGGCGGCCT
CAAGCACGGC
CCACAAGTTC
CAACCTGTGC
CTTCATGTAC
GAACAGCTGC
GTGCATCTGC
CAAGTTCTAC
CTGGGAGCCC
CGTGTCCATG
GTACAAGGCC
GACCCGCGAG
CGCCAGCGGC
AGGTCATAAT
cagcagctgg
ctgattgata
ggtgaccagg
aggtaggcac
gttagcgtgt
tactggcaat
cgtgcctgcg
tttgcaagga
tggacggacg
tgcaatgcgg
gcgctcactg
taatcgcctt
cgatcgccct
cgcattaagc
cctagcgccc
ccgtcaagct
cgaccccaaa
ggtttttcgc
tggaacaaca
ttcggcctat
aatattaacg
tgtttatttt
atgcttcaat
attccctttt
gtaaaagatg
agcggtaaga
aaagttctgc
cgccgcatac
cttacggatg
actgcggcca
cacaacatgg
ataccaaacg
ctattaactg
gcggataaag
gataaatctg
ggtaagccct
cgaaatagac
III
C
CTTGTGGTGG
CCCAGCAAGG
GTGGGCAAGA
CTGACCAAGG
GTGATCACCG
GTGGTGGAGG
GGCAACCGCG
CCCGCCGGCT
AACGCCGACA
GGCGTGAACT
AGCTGCGAGA
TACCTGCTGC
AAGAGCGTGC
GACCGCAGCG
AGCGCCCTGC
CATAATCATA
accgcctgta
cgggatcgga
gtcggtgtgg
gtcgacttgg
gcgtgagcca
atctgccaat
cgcgccccgg
cagggtaatc
gcaggggagg
ccgccaccgc
gccgtcgttt
gcagcacatc
tcccaacagt
gcggcgggtg
gctcctttcg
ctaaatcggg
aaacttgatt
cctttgacgt
ctcaacccta
tggttaaaaa
cttacaattt
tctaaataca
aatattgaaa
ttgcggcatt
ctgaagatca
tccttgagag
tatgtggcgc
actattctca
gcatgacagt
acttacttct
gggatcatgt
acgagcgtga
gcgaactact
ttgcaggacc
gagccggtga
cccgtatcgt
agatcgctga
ATATGGACGA
TGGGTGTGGT
GCGCCGCCGC
GCAACGCGGG
AGATGACCAT
GCGAGGGCAT
GCGGCCCCCT
TGTTCACCGA
ACACCTGGGA
TCACCGTGTC
TCCCCGCCGA
AGATCATCCC
TGAAGGACGG
CCCGCAAGAT
ACGCCAAGAA
CCGGCGGCCG
ATCATAATCA
ccatggagaa
gctcggaggc
ggtcggccca
tttgcgaccc
gtggccaacg
gccatactgc
gggcgcagtt
acagcagcaa
gacggcgcag
ggtggagctc
tacaacgtcg
cccctttcgc
tgcgcagcct
tggtggttac
ctttcttccc
ggctcccttt
agggtgatgg
tggagtccac
tctcggtcta
atgagctgat
aggtggcact
ttcaaatatg
aaggaagagt
ttgccttcct
gttgggtgca
ttttcgcccc
ggtattatcc
gaatgacttg
aagagaatta
gacaacgatc
aactcgcctt
caccacgatg
tactctagct
acttctgcgc
gcgtgggtct
agttatctac
gataggtgcc
GGGAGACGCG
CCTGCTGGAC
CGCGGATCGG
GCTGTGGGAG
GAAGTACCGC
CGGCTACCCC
GCCCTTCGCC
GTACCCCCAG
CCGCTCGTTC
CGTGGAGGAG
CGGCCCCGTG
CGTGCCCAAG
CGGTCGCCTG
GCCCGACTGG
CCAGAAGTGG
CCTGGTTCCG
TAATCACAAT
gagctttact
tttcgcgcta
cggtcaatta
cgcagttttg
tgccacaccc
atgtaatggc
tagctgacca
catggtgggc
ctcgggagac
caattcgccc
tgactgggaa
cagctggcgt
gaatggcgaa
gcgcagcgtg
ttcctttctc
agggttccga
ttcacgtagt
gttctttaat
ttcttttgat
ttaacaaaaa
tttcggggaa
tatccgctca
atgagtattc
gtttttgctc
cgagtgggtt
gaagaacgtt
cgtattgacg
gttgagtact
tgcagtgctg
ggaggaccga
gatcgttggg
cctgtagcaa
tcccggcaac
tcggcccttc
cgcggtatca
acgacgggga
tcactgatta
Appendixes
3202
3262
3322
3382
3442
3502
3562
3622
3682
3742
3802
3862
3922
3982
4042
4102
4162
4222
4282
4342
4402
4462
4522
4582
4642
4702
4762
4822
4882
4942
5002
5062
5122
5182
5242
5302
5362
5422
5482
5542
5602
5662
5722
5782
5842
5902
5962
6022
6082
6142
6202
6262
6322
6382
6442
6502
6562
6622
6682
6742
6802
agcattggta
atttttaatt
cttaacgtga
cttgagatcc
cagcggtggt
tcagcagagc
tcaagaactc
ctgccagtgg
aggcgcagcg
cctacaccga
ggagaaaggc
agcttccagg
ttgagcgtcg
acgcggcctt
cgttatcccc
gccgcagccg
tacgcaaacc
ttcccgactg
aggcacccca
gataacaatt
ctcactaaag
aaagaggcca
ttttacgtgt
cttgagagca
gcgcctccat
cgcaacccta
gtaaaacgcc
gaacgccgcg
caccagcgcg
ggtcacctcg
aaccgccaga
cgaccacccc
acgggccgcc
cagcgcgtgc
ccgcggccac
cctctcgtcc
caccagccgc
aaacaactcc
cacaacaacc
gcttcgaaat
acgcagggtt
atgcgggcgt
gactcacctg
ggccccctgg
attggcgcct
accccatcaa
ttgatgggat
taagtgctcg
ggatgggtgc
ccttccagag
catacgcacc
ctgcctgaca
cgtcagaaac
cctttcctag
gtgccagcgg
gagcaggcgg
cgcctccaca
gtccttaaaa
ctttcctgac
caaatctggc
cgtgattatt
actgtcagac
taaaaggatc
gttttcgttc
tttttttctg
ttgtttgccg
gcagatacca
tgtagcaccg
cgataagtcg
gtcgggctga
actgagatac
ggacaggtat
gggaaacgcc
atttttgtga
tttacggttc
tgattctgtg
aacgaccgag
gcctctcccc
gaaagcgggc
ggctttacac
tcacacagga
ggaacaaaag
aaatcaacgg
tgaaaaagac
gtatcttcca
ttacacggag
cccagccacc
agcttttcct
gaacactccg
agatcggagt
caggtacgag
tcctcgtccg
ttccgctcct
tgcggcaccg
agcgaacgag
cgcgcactgg
ccaccaccct
tcagcctcac
cgcccaccac
cactcacaac
tcttcagcac
agatgctgct
tgcaagtcaa
gccattttaa
ccggtttatc
agcctttgtg
acatcatcct
cttaagctag
tttgtcgctg
cgccagcggg
cttcccgcgc
aatcatgtca
ggaagtgaac
acgtctccgc
gccaccgaca
tgcccttgtg
cctggctgtt
cctaccgatg
agccgcttct
gcgctcggcc
gaggcagtag
ggtatttacg
caagtttact
taggtgaaga
cactgagcgt
cgcgtaatct
gatcaagagc
aatactgtcc
cctacatacc
tgtcttaccg
acggggggtt
ctacagcgtg
ccggtaagcg
tggtatcttt
tgctcgtcag
ctggcctttt
gataaccgta
cgcagcgagt
gcgcgttggc
agtgagcgca
tttatgcttc
aacagctatg
ctgggtaccc
aggatcgtta
tgatcagcac
tccaccgccg
cggggatccc
aacaccatca
ccgataccgc
gcccgaacca
gccggtccgc
ggtcgagcag
caggccgagt
cgtccagatc
tcaccgcgag
cgagccccgc
ccggacgccc
ccacaacacg
ccaacaagcc
cccgaagccg
cgggataccg
cggggagggc
tgagacagcg
atctgcaagc
gatgttgagt
aggagggcac
ggccaggggg
ggtttggctg
ctgagtggtt
aaagtggagg
ccgtatggcg
cctcatagcc
agcctcagcg
gcatgtcgag
cacgctctcc
ggacccaggc
ctggtgatcg
cgaagggctc
cgtgaaggca
tgtcgtcgtt
ccatattcgg
gcttttaagt
cgacggcccg
IV
catatatact
tcctttttga
cagaccccgt
gctgcttgca
taccaactct
ttctagtgta
tcgctctgct
ggttggactc
cgtgcacaca
agctatgaga
gcagggtcgg
atagtcctgt
gggggcggag
gctggccttt
ttaccgcctt
cagtgagcga
cgattcatta
acgcaattaa
cggctcgtat
accatgatta
gcttcaaata
caaccaacaa
gaaacgggga
ttcgtcaggg
aacgtccaca
ggtccctcag
cccatcccac
cgggtcctcc
acggccgacc
cacgttgtcc
ccgctccagc
ctccaagtcg
actgcgatcg
gagcgccacc
cggaaccgct
aggtacggga
caccccggcc
ataaacacca
accccgcagt
ggagtggcca
acagaggagc
acgctgcctg
gacttctctt
cgctccaggg
cttccggata
cgctccttct
atgtatagcg
tcaccgttcg
ccttctggac
cgccaaatca
agctccccgc
ggaggcctca
ctctcacggc
gctctcagca
cttggaagcg
gccgccagtt
ggcaaatgct
ccgagacatg
acgcaattgt
tgcaaggcga
gcgcgttagc
ttagattgat
taatctcatg
agaaaagatc
aacaaaaaaa
ttttccgaag
gccgtagtta
aatcctgtta
aagacgatag
gcccagcttg
aagcgccacg
aacaggagag
cgggtttcgc
cctatggaaa
tgctcacatg
tgagtgagct
ggaagcggaa
atgcagctgg
tgtgagttag
gttgtgtgga
cgccaagcgc
cgcccagccc
aattgcaaaa
gctaagctac
ggcaaggctc
ctgtgctgtc
aagaactcgt
ccgcgcccgt
tcgtgggcca
cgccccacgt
gggcacaggt
tcggcgagaa
acgctccctt
aacggacacc
gccacgtcca
tcggtgacca
atccccacct
cccagagctg
gcccccgagg
gcacgcaacg
tcctgcaaat
caaaagcctt
atccgccggg
gtaaaaaagt
gctgcatgcg
agggttgcaa
ggcgcgcccg
gcagaatagt
gggtcgcggg
gccgcgcgcc
gtcctgtagc
tcgagcacac
ccaatcgtca
cgaccccgca
tgcctcaaca
catgcgaaga
cgggtgcctt
catgtttgcc
ttagcagatc
catttgtagc
gagagcaaag
ggcccttccc
ttaaaacttc
accaaaatcc
aaaggatctt
ccaccgctac
gtaactggct
ggccaccact
ccagtggctg
ttaccggata
gagcgaacga
cttcccgaag
cgcacgaggg
cacctctgac
aacgccagca
ttctttcctg
gataccgctc
gagcgcccaa
cacgacaggt
ctcactcatt
attgtgagcg
gcaattaacc
gcccatggag
ctcctccgct
cgcttcagca
agatcaacga
acccacgcga
ccaacagccg
actcccgcag
gctcgcgcag
cgatcagccc
gaccgtggca
gccgctcccc
cagcgacagc
gctcccagtc
gccgctgctc
accaggcgac
ccgccaacca
ccaccttgac
ccccatcctc
catcgtccat
ggaaacggcg
cgtcgacaca
cttgctcgtc
aaagaacata
aactgcttgc
gtgctcaaat
gcatgcaagc
cgcgtatgta
cttttatacc
ccatcgcggc
ttcatacaaa
acctgcccgt
cacgagccct
gcccttttgc
acccgtactc
cgaaggggcg
tctccacgcg
cgaactcgga
gcagtgccac
acaattggag
tgggacgcgg
ccaggccagg
Appendixes
6862
6922
6982
7042
gacgattatg
ggagggggat
cgccgagcaa
ctgctactca
tatcaatatt
ctgggaattg
gccagggtta
caacaagccc
gttgcgttcg ggcactcgtg cgagggctcc tgcgggctgg
gaggtacgac cgagatggct tgctcggggg gaggtttcct
ggtgttgcgc tcttgactcg ttgtgcattc taggacccca
a
Features:
Purple-5’rppH (4..210).
Green-GFP (210..906).
Light Blue-Histidine tag (946..982).
Orange-3’PSAD terminator region (994..1379).
Dark blue- HSP70A-RBCS2_promoter::aphVIII::RBCS2_terminator (4430..6241).
Red-5’PSAD promoter region (6242..7062).
Double Underlined-Different primers used .
Single Underlined-Ndel/Xbal/Notl/Xhol/SmaI/EagI restriction sites.
TT-The 5’ and 3’ end of second and first primers in their respective strand.
V
Appendixes
APPENDIX IV
Codon Usage Table
(Nakamura et al. 2000).
VI
Appendixes
APPENDIX V
Primer List:
Genomic DNA amplification:
PCR at annealing temperature of 63 ˚C
5’PSAD6401 : 5’- CGCCGAGCAAGCCAGGGTTA-3’ Tm (63.5˚C)
3’PSAD495 : 5’-GCGAAAGCCTCCGAGCTCCGAT-3’ Tm (65.8˚C)
PCR at annealing temperature of 63.5 ˚C
NUD3’-5COMP: 5’-TTCGGCTGCTTGTGGTGGTGGG-3’ Tm (54˚C)
3’GFP: 5’-GTCATCTCCTTGGTCAGGCCG-3’ Tm (64 ˚C)
PCR at annealing temperature of 61 ˚C
5’paro4331:5’-ACGGCCGACCCGCCCCACGT-3’ Tm (69.6 ˚C)
3’paro4743-:5’-GATTCCCGTACCTCGTGTTGT-3’ Tm (59.8 ˚C)
PCR at annealing temperature of 61 ˚C
5’NUD GFP:5’-CTGCCGCCCCTGTGGCGTT-3’ Tm (67.6 ˚C)
3’ PSAD 495: 5’-GCGAAAGCCTCCGAGCTCCGAT-3’ Tm (65.8˚C)
Plasmid DNA amplification:
PCR at annealing temperature of 65 ˚C, pBluescriptII SK+
5'NUD-22 Forward : 5’-ACCTTTGCCACCATGGACGAG-3’
5'NUD-22 Reverse : 5’-GGAAACAGCTATGACCATG-3’
PCR PCR at annealing temperature of 65 ˚C ,NcoI→NdeI
Forward : 5’-AAGAAGGAGATATACTATGGACGA-3’
5’-AAGAAGGAGATATAATATGGACGA-3’
5’-AAGAAGGAGATATCATATGGACGA-3’
Reverse : 3’-CCAACTCAGCTTCCTCTAGAT-5’
VII
Appendixes
APPENDIX VI
Abbreviations:
Ampr-Ampiciline resistant
AphVIII- Aminoglycoside 3'-phosphotransferase type VIII
APS- Ammonium persulfate
MCS-Multiple cloning sites
NEP- Nuclear-encoded plastid RNA polymerase
ORF-Open reading frame
PEP- Plastid encoded RNA polymerase
PPR- Pentatricopeptide repeat proteins
RBCS2-HSP 70- Ribulose bisphosphate carboxylase small chain 2-Heat shock prtein 70
Rpm-Rounds per minute
SDS-PAGE-Sodium dodecyl sulfate polyacrylamide gel electrophoresis
TAE- Tris-acetate-EDTA
UTR- Untranslated region
VIII
Appendixes
APPENDIX VII
Bioinformatic tools:
C.U.D (http //www.kazusa.or.jp/codon)
Expasy (http://www.expasy.org/)
GCUA (http://gcua.schoedl.de/)
KEGG pathway (http://www.genome.jp/kegg/pathway.html)
NCBI (http://www.ncbi.nlm.nih.gov/)
NEB cutter V2.0 (http://tools.neb.com/NEBcutter2/)
Uniprot (http://www.uniprot.org/)
IX